Electrospray ionization mass spectrometry (ESI/MS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), tandem mass spectrometry (MS/MS), matrix-assisted laser desorption ionization (MALDI) mass spectrometry, accurate mass measurement for elemental composition determination, and combined capillary electrophoresis (CE) and mass spectrometry remain the techniques of current interest. Because the low-molecular-weight matrices used for conventional MALDI/MS are in 100- to 1000-fold excess to the sample, the ions from the matrix often obscure the lower mass range of the spectrum and make the analysis of small molecules (molecular weight range = 200-1000) problematic. We have found that the best overall conventional matrix for small molecules is 3,4-dihydroxybenzoic acid (DHB), although it is not perfect or applicable to every compound. MALDI/MS using special energy-transferring surfaces and direct laser-desorption ionization (LDI) without the presence of a matrix both show substantial promise for overcoming matrix limitations and facilitating the rapid analysis of small molecules . LDI mass spectra could be obtained from a wide variety of molecular structures including those compounds that would not be expected to readily absorb energy from a UV laser. Initial studies have shown that LDI/MS is comparable to conventional MALDI/MS in its ability to characterize small molecules. Unconventional MALDI matrices (e.g. graphite) have also been investigated in this study. MALDI spectra obtained using graphite from pencil lead exhibit extensive alkali metal ion cationization and show substantial variation depending on the source of the pencil lead. However, the best performing graphite matrix appears to be both comparable and complementary to conventional MALDI/MS in its ability to characterize small molecules. A more comprehensive evaluation employing blind control compounds is ongoing with the goal of choosing the MALDI or LDI methods that give the best chance of providing unambiguous molecular weight information. In addition, the off-line combination of MALDI/MS with high-performance liquid chromatography (HPLC) is under investigation as an alternate approach for the targeted analysis of complex biological and synthetic mixtures. This approach involves automatically collecting and spotting the HPLC eluent along with an appropriate matrix on a MALDI sample plate for subsequent MS analysis, which can include accurate mass measurement and post-source decay fragmentation analysis or MS/MS. An advantage of this approach over on-line LC/MS is the generation of an archive of the original separated sample for re-analysis. Synthetic derivatives built on a constrained diacylated glycerol scaffold (DAG-lactones) are potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents corresponding to the acyl groups R1 and R2, these DAG-lactones exhibit varying degrees of PK-C isozyme specificity and produce a multitude of unique biological activities. A solid-phase combinatorial approach has been applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. We have applied a variety of mass spectral analysis strategies to rapidly characterize these synthetic libraries before biological evaluation. Flow-injection APCI/MS of individual DAG-lactones produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions. This later approach, especially if MS/MS techniques are used, appears to be complementary to FAB/MS in terms of spectral information, although it is not suitable for mixture analysis. However, flow-injection APCI/MS is amenable to automation and allows multiple analyses of the same sample to be carried out unattended. MALDI/MS also appears useful as a tool for the rapid characterization of these small molecule libraries, especially when a pencil lead graphite matrix is used (see above). Optimization of matrix and automation of the sample deposition process are under investigation as a rapid and routine analytical screening method. Our goal is the comprehensive structural characterization of all 96 library components in one day.